The present invention relates to methods of screening subjects for mitochondrial dysfunction.
A wide variety of clinical manifestations are due to mutations in mitochondrial DNA, but are difficult to diagnose due to the varied clinical picture and the lack of sensitive or specific diagnostic testing. Past efforts to document mtDNA mutations in children believed to have mitochondrial disorders have been hampered by the size of the mitochondrial genome and the presence of numerous benign polymorphisms.
Mitochondria are eukaryotic cytoplasmic organelles where oxidative phosphorylation takes place, and are often termed the xe2x80x98power plantxe2x80x99 of the cell. In animal cells, the mitochondria is the only cytoplasmic organelle that contains DNA. Human mitochondrial DNA (mtDNA) is a circular molecule of about 16,600 nucleotide pairs which encode thirteen of the at least 82 protein subunits of the complexes in the oxidative phosphorylation pathway, both ribosomal RNAs, and all of the 22 transfer RNAs required for mitochondrial protein synthesis. However, the majority of proteins located in the mitochondria are encoded by nuclear DNA (chromosomal DNA) and translated by cytoplasmic ribosomes, and then imported to the mitochondria. Therefore, a xe2x80x9cmitochondrial disorderxe2x80x9d can be secondary to a mutation in either the nuclear DNA or in the mitochondrial DNA.
The entire human mitochondrial DNA (mtDNA) sequence has been determined (see MITOMAP: Human Mitochondrial Genome Database, Center for Molecular Medicine, Emory University, Atlanta, Ga., USA (1998); Wallace et al. (1995) Report of the committee on human mitochondrial DNA, In: Cuticchia A J (Ed) Human gene mapping 1995: A Compendium, Johns Hopkins University Press, Baltimore, pp 910-954 (1995)).
Mitochondrial genetics differ from nuclear (standard or Mendelian) genetics. Virtually all the mtDNA of a zygote is derived from the oocyte, and mtDNA disorders are transmitted by maternal inheritance. Maternal-linked (matrilineal) relatives presumably have identical mtDNA sequences, except perhaps at the site of a new mutation. Additionally, the mtDNA mutation rate is substantially higher than that of the nuclear DNA. Most cells contain hundreds to thousands of mitochondria, and each mitochondria contains several copies of mtDNA, resulting in high mtDNA copy number. In normal individuals, essentially all of the mtDNA molecules are identical (homoplasmy). However, if there is a mutation in mtDNA, the mutant mtDNA and the normal (wild type) mtDNA often coexist in the same cell or tissue (heteroplasmy).
Because of the high mutation rate, mtDNA has numerous polymorphisms. Almost always these polymorphisms are homoplasmic. In contrast, most recognized pathogenic mtDNA mutations are heteroplasmic, especially when disease manifests during childhood (Shoffner and Wallace (1995) In: The Metabolic and Molecular Basis of Inherited Disease (7th Ed.), New York, McGraw Hill, 1535-1629).
As a result of segregation in the pre-oocyte stage, each ova of an affected woman has a different proportion of mutant versus normal mtDNA, which can range from virtually 0 to 100%. Each of her children, therefore, will inherit differing amounts of mutant mtDNA. In addition, normal and mutant mtDNA randomly segregate during the cell divisions of embryogenesis, resulting in different proportions of mutant mtDNA residing in different tissues. The presence of clinical disease in a given tissue is dependent on the specific mutation, the percent of mutant mtDNA and the threshold for that tissue. The percentage of mutant mtDNA necessary to cause clinical symptoms varies from tissue to tissue; for example, 80% mutant mtDNA may be clinically silent in liver but cause symptoms in tissues with higher energy requirements, such as muscle or brain (Shoffner et al. (1991) Adv. Hum. Genet. 19:267). Since the mutant mtDNA load varies between matrilineal family members, as well as between tissues within each individual, the clinical manifestations of a mtDNA mutation vary widely among affected family members. Healthy family members with mutant mtDNA levels below threshold are common. These individuals, if female, are xe2x80x98carriersxe2x80x99 as their children will inherit their mitochondria and, if inherited mutant mtDNA levels are above threshold, the children will be affected. A well known example is the A3243G mtDNA mutation, in which family subjects exhibit variable manifestations, ranging from stroke (usually associated with relatively higher degrees of mutant heteroplasmy) to those (with lesser mutant loads) with diabetes, deafness, or asymptomatic carriers. This phenomenon of varied clinical presentation has been observed with other mtDNA mutations as well.
As the mtDNA mutation rate is high, mtDNA disorders may be due to new mtDNA mutations; in such cases matrilineal relatives will be unaffected. In other cases, mothers harbor small degrees of mutant heteroplasmy and are clinically normal or only mildly affected. In a minority of cases, multiple matrilineal relatives harbor various amounts of mutant mtDNA in their tissues and exhibit varying clinical manifestations.
A broad spectrum of disease manifestations has been associated with systemic mtDNA mutations. These mutations can be either single point mutations, or large rearrangements (deletions and/or duplications). Rearrangements usually are spontaneous, although they may be maternally inherited or mendelianly inherited secondary to predisposing nuclear mutations.
Clinical mitochondrial dysfunction may be defined as idiopathic neuromuscular and/or multisystem disease, biochemical signs of energy depletion, and lack of another diagnosis. Mitochondrial disorders are evidenced when the cellular supply of energy is unable to keep up with demand; symptoms predominate in tissues with the highest energy requirements (brain and muscle). Mitochondrial disorders are most commonly displayed as neuromuscular disorders, including developmental delay, seizure disorders, hypotonia, skeletal muscle weakness and cardiomyopathy. Other manifestations which have been reported include gastroesophageal reflux, apnea, optic atrophy, deafness, acute liver failure, diabetes mellitus, and other hormonal deficiencies.
Mitochondrial disorders are often not suspected until late in a diagnostic work-up. Confirmation of a mitochondrial disorder is, at present, a time-consuming and expensive process, and may include lactic acid measurement in body fluids and diagnostic muscle biopsy for electron microscopy and assay of the electron transport chain activities in vitro. However, these methods rarely specify the mode of inheritance or allow for presymptomatic or prenatal diagnosis.
A method of screening a subject for mitochondrial dysfunction is disclosed. The method comprises detecting the presence or absence of single nucleotide changes in a hypervariable region of the mitochondrial DNA of said subject, the presence of such changes indicating that said subject is afflicted with or at risk of developing mitochondrial dysfunction.
Also disclosed is the use of a means for detecting the presence or absence of single nucleotide changes in a hypervariable region of the mitochondrial DNA of a subject in or for determining if that subject is afflicted with or at risk of developing mitochondrial dysfunction.
The foregoing and other objects and aspects of the present invention are explained in greater detail below.